In humans, vision is the most important sense and damage to the retina or the optic nerve can cause irreversible vision loss. This is because the retina and the optic nerve are part of the central nervous system (CNS), which in adult mammals has lost its regenerative capacity. In contrast, amphibians and fish, including zebrafish, have retained a remarkable capacity to generate new CNS neurons, and to re-grow severed or damage axons and nerves, including the optic nerve. The optic nerve, which conveys visual information from the retina into the brain contains axons from only one retinal cell type, the retinal ganglion cells (RGC). After optic nerve transection, zebrafish RGC neurons survive, and -independently of neurogenesis- regrow axons to their original synaptic targets where they form functional synapses. Surprisingly, the molecular genetic pathways for this remarkable capacity to regenerate axons in vivo, are not well understood. Here, I propose to take advantage of this regenerative capacity and to perform a genetic and a small molecule screens to identify genes and pathways required for optic nerve regeneration in vivo. We have chosen this system because it is very accessible to simple, rapid and reproducible nerve transection amiable to screens, because of our longstanding interest and expertise in visual system development and function, and because all findings regarding regeneration in the visual are also translatable to CNS regeneration in general. The long term-goal of this research proposal is to define the genetic, molecular and cellular pathways underlying axonal regeneration. The experiments in this proposal will: (1) screen an equivalent of ~970 chemically mutagenized genomes for defects specifically in optic nerve axonal regeneration; (2) identify the molecular nature of at least 30 mutants (through a whole genome sequencing approach); and (3) perform a small molecule pilot screen of 1760 small molecules with known targets to identify factors that delay axon fragmentation, and de- or increase optic nerve axonal regeneration to define entry points into pathways underlying axonal regeneration. These studies are relevant to the study of human diseases that cause damage to the optic nerve, including hereditary optic neuropathies, cancer or multiple sclerosis as well conditions of increased intraocular pressure which can cause irreversible optic nerve degeneration and vision loss. Given the current lack of therapeutic interventions for optic nerve damage or for spinal cord injury in general, we propose to apply a more unbiased genetic approach to determine the molecular mechanisms underlying axonal regeneration, and to exploit these mechanisms towards the development of therapeutic strategies in mammals.